PROJECT SUMMARY This study aims to delineate key molecular mechanisms driving the most common genetic cause of amyotrophic lateral sclerosis (ALS) pathogenesis. ALS is a lethal neurodegenerative disorder most commonly associated with the accumulation of arginine-rich dipeptide repeats (DPRs) in membrane-less organelles which leads to the death of motor neurons. Arginine-rich DPRs infiltrate nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter NPM1 in vitro liquid-liquid phase separation. NPM1 is crucial to the maintenance of nucleolar liquid-like properties through its ability to phase separate with proteins and nucleic acids. How interactions with NPM1 enable ALS-related DPRs to accumulate in nucleoli and induce nucleolar dysfunction/motor neuron cell death remain unknown. Elucidating the mechanisms and effects of DPR interactions with NPM1 on the liquid-like properties, function, and architecture of nucleoli that lead to nucleolar dysfunction and cell death is critical for understanding and treating ALS. Two mechanistic hypotheses will be tested. First, DPRs outcompete nucleolar proteins in binding to NPM1, forming DPR-saturated complexes that disrupt NPM1-mediated phase separation, and thus perturb nucleolar function and induce toxicity. NPM1 undergoes in vitro phase separation with nucleolar proteins. DPRs can insinuate into these liquid-like protein droplets and dissolve them. I will be trained in confocal fluorescence microscopy and NMR techniques which will enable me to elucidate the mechanism of NPM1/DPR interactions. I will combine training in analytical ultracentrifugation, fluorescence, and neutron scattering methods with my expertise in light and x-ray scattering to determine the mechanisms of DPR competition with different nucleolar proteins in binding NPM1 and the sequestration of NPM1 into ?phase-separation inhibited? complexes. Second, NPM1 phase separation thresholds are lowered, and NPM1/DPR phase separated droplets become more gel-like, with DPR length-dependent potency. I will establish the DPR length-dependence of in vitro NPM1-containing droplet formation/dissolution and rigidification by quantitative scattering/turbidity and confocal microscopy analyses. In cell models of ALS, I will be trained to use NPM1 localization and mobility, nucleolar stress, and cell toxicity assays to further test the hypotheses developed from biophysical studies. This proposal aims to bridge the gap between observed DPR burden in brains and cellular toxicity via the alteration of nucleolar phase separation by NPM1. The insights obtained from the proposed studies can be used to (1) further our understanding of the molecular determinants driving biomolecular phase separation in complex systems, (2) drive a systems level approach to the study of phase separation mechanisms in ALS and other disorders, and (3) develop therapeutics specific to ALS, targeted to NPM1 phase separated systems, and/or targeted to phase separated systems in general (e.g., by promoting or targeting specific states/complexes).